The role of biotin in regulating 3-methylcrotonyl-coenzyme a carboxylase expression in Arabidopsis

Abstract

As a catalytic cofactor, biotin has a critical role in the enzymological mechanism of a number of enzymes that are essential in both catabolic and anabolic metabolic processes. In this study we demonstrate that biotin has additional non-catalytic functions in regulating gene expression in plants, which are biotin autotrophic organisms. Biotin controls expression of the biotin-containing enzyme, methylcrotonyl-coenzyme A (CoA) carboxylase by modulating the transcriptional, translational and/or posttranslational regulation of the expression of this enzyme. The bio1 mutant of Arabidopsis, which is blocked in the de novo biosynthesis of biotin, was used to experimentally alter the biotin status of this organism. In response to the bio1-associated depletion of biotin, the normally biotinylated A-subunit of methylcrotonyl-CoA carboxylase (MCCase) accumulates in its inactive apo-form, and both MCCase subunits hyperaccumulate. This hyperaccumulation occurs because the translation of each subunit mRNA is enhanced and/or because the each protein subunit becomes more stable. In addition, biotin affects the accumulation of distinct charge isoforms of MCCase. In contrast, in response to metabolic signals arising from the alteration in the carbon status of the organism, biotin modulates the transcription of the MCCase genes. These experiments reveal that in addition to its catalytic role as an enzyme cofactor, biotin has multiple roles in regulating gene expression.

Figure 1

The effect of plant growth on MCCase activity. MCCase specific activity was determined in extracts of wild-type or bio1 mutant seedlings between 3 and 28 d after sowing. Seedlings were grown either with or without the exogenous addition of 1 mm biotin. Data are the mean ± se from four replicates.

Figure 2

The effect of biotin on the biotinylation status and accumulation of the MCCase subunits. Protein extracts were prepared from seedlings (A–C) or excised cotyledons (D) of wild-type and bio1 Arabidopsis seedlings at the indicated DAP. Aliquots of extracts containing equal amounts of protein (150 μg) were subjected to SDS-PAGE, followed by western-blot analysis with either ¹²⁵I-streptavidin to detect the biotinylated MCC-A subunit (A) or immunological detection with antibodies to MCC-A (B) or MCC-B (C and D). Where indicated, exogenous biotin (0.25 mm) was provided to the bio1 seedlings 2 d before harvest. The data presented were gathered from a single experiment; five replicates of this experiment, with two different batches of bio1 seeds, gave similar results.

Figure 3

The effect of biotin-depletion on MCCase gene transcription. Northern-blot analysis of MCC-A (A) and MCC-B (B) mRNA accumulation in wild-type and bio1 Arabidopsis seedlings. RNA was isolated from wild-type and bio1 seedlings grown to 20 DAP in the absence of exogenous biotin. Equal amounts of isolated RNA (50 μg) were subjected to electrophoresis in formaldehyde-containing agarose gels, and MCC-A or MCC-B mRNAs were detected by hybridization with respective ³²P-labeled probes. Reporter gene expression studies of the MCC-A and MCC-B genes. GUS activity was determined in protein extracts from transgenic Arabidopsis seedlings of either wild-type (wt) or bio1 genetic background and carrying an MCC-A::GUS (C) or MCC-B::GUS (D) reporter transgene. Seedlings were grown without exogenous biotin to the indicated DAP. Data are the means ± se from three replicates.

Figure 4

The effect of biotin-depletion on the metabolic regulation of MCC-A and MCC-B gene transcription. GUS activity was determined in protein extracts from wild-type (wt) or bio1 Arabidopsis seedling carrying an MCC-A::GUS (A) or MCC-B::GUS (B) reporter transgene. Seedlings were grown to 13 DAP on Murashige and Skoog agar medium without biotin, followed by 2 d of additional growth either in the absence (−) or presence (+) of exogenous biotin. In these last 2 d of growth, seedlings were grown either under constant illumination (white bars), or transferred to darkness (black bars), or CO₂-free air (dotted bars). Data are the means ± se from three replicates.

Figure 5

Time course of the biotin dependence of the metabolic regulation of MCC-A and MCC-B gene transcription. GUS activity was determined in protein extracts from wild-type (wt) or bio1 Arabidopsis seedlings carrying an MCC-A::GUS (A) or MCC-B::GUS (B) reporter transgene. Seedlings were grown in the absence of exogenous biotin to the indicated DAP. These seedlings were maintained in constant illumination until the last 2 d of growth, at which stage they were transferred to total darkness. Data are the means ± se from three replicates.

Figure 6

Electrophoretic characterization of MCCase. Protein extracts from Arabidopsis, soybean, and pea seedlings (A) and wild-type (wt) and bio1 mutant Arabidopsis seedlings (B) were subjected to exhaustive electrophoresis (for 14,400 V h⁻¹) in gels composed of a linear gradient of 5% to 30% polyacrylamide according to the method of Hedrick and Smith (1968). After western blotting, MCCase was immunologically detected by reacting the membranes with anti-MCC-B serum (identical results were obtained with anti-MCC-A serum; data not shown). The native molecular mass of MCCase was determined by comparing its migration to standard proteins (apoferritin dimer, 886 kD; apoferritin monomer, 443 kD; urease dimer, 545 kD; and urease monomer, 272 kD). The position of MCCase is indicated by arrows. C, Analysis of MCCase charge isoforms. Aliquots of protein extracts from wild-type (wt) and bio1 Arabidopsis seedlings at the indicated DAP, containing equal amounts of MCCase activity, were subjected to electrophoresis at 70 V for 17 h in a linear 5% to 20% gradient polyacrylamide gel (Lambin and Fine, 1979). After western blotting, MCCase was immunologically detected by reacting the membranes with anti-MCC-B serum (identical results were obtained with anti-MCC-A serum; data not shown).